pGEM-gbr22 Protein Expression, Purification and Characterization through E.Coli
Introduction:
The process of obtaining protein for use in biochemical assays or antibody production involve protein expression and purification techniques [1]. The DNA coding for the protein must first be incorporated into a host cell which can range from bacterial to yeast cells. When using BL-21 bacterial E. Coli, a single recombinant colony is grown into a starter culture which is induced into protein expression and finally harvested for the target protein. Expression may be optimized by use of a tag (e.g. hexa-histidine affinity tag) or by varying expression induction conditions such as temperature and concentration [2]
The goal of protein purification is to isolate the protein. A clarified sample of soluble protein is first extracted via centrifuge and then clarified further by removing DNA and lipids. For His6-tags, a combination of imidazole buffer, phosphate buffer, and Ni-NTA resin is then used to bind and extract the protein [3].The success of expression and purification then can be gauged via gel electrophoresis and UV-vis spectroscopy.
Each individual protein has unique expression and purification protocols [1]. In this lab, gbr22, a purple protein found in coral, is expressed, purified, and characterized to determine the extent of purification. By utilizing appropriate expression and purification technique, characterization through gel electrophoresis should yield a molecular weight of gbr22 similar to the literature value as well as a high concentration of purified protein.
Materials & Methods:
gbr22 protein in the form of a plasmid with antibiotic resistance was mixed with a culture of bacteria and heat shocked to initiate transformation. The bacteria culture was then spread onto antibiotic plates incubated overnight at 37OC to yield purple colonies of living transformed bacteria. A starter culture was then grown with a single purple colony, LB media, and 100 mg/ml ampicillin and put into a shake incubator at 37OC for ~8 hours. The starter culture was then transferred into a larger culture with additional ampicillin and LB and shake incubated for 24 hours (sample 1 taken of large culture). The purple culture was then spun down to obtain a purple pellet of transformed cells. 1X PBS and 1 mg/mL lysozyme were added to the pellet and the final tube was frozen at -20OC.
The lysate was purified by benzonase to digest DNA, centrifuge (supernatant was taken as sample 2 at this point), and finally PES syringe filter of the clarified liquid lysate. The mixture was further purified via Ni-NTA affinity through a Bio-rad Econo column. 1X PBS, 20 mM imidazole, and 250 mM imidazole was run through the column to produce waste, wash, and two elutions. Samples 3, 4, 5, and 6 were taken of these steps, respectively. The Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) was then used to measure the absorbance of the first elution at 280 nm and 574 nm (the maximum absorbance wavelength of gbr-22).
In order to characterize the purified protein, gel electrophoresis was conducted with SDS-PAGE gel along with six samples taken at various points in the expression and purification steps (specified in the results section). The gel results were then compared to a molecular weight standard.
Results:
Figure 1: Top: BL-21 plate with pGEM-gbr22 plasmid – purple colonies (a few thousand) are transformed bacteria. Bottom: BL-21 plate without plasmid – no purple colonies.
Figure 2: Purple culture of a single BL-21 colony transformed with pGEM-gbr22 incubated for 20 hours in 100 g/ml ampicillin and LB media.
Figure 3: Purple transformed BL-21 cell pellet isolated by centrifuge (5000 rpm for 10 minutes at 4OC). Mass of purple pellet = 0.4700 ± 0.0002 g.
Figure 4: Elutions 1 (left) and 2 (right) containing purified gbr22 protein released from Ni-NTA resin using 250 mM imidazole and 1 M PBS solution.
Figure 5: Nanodrop spectrophotometer (Protein A280 mode) results for n=2 trials of Elution 1 containing purified gbr22 protein using 280 nm. Absorbance: 0.541 for n=1, and 0.535 for n=2.
Figure 6: Nanodrop spectrophotometer (UV-vis mode) results for n=2 trials of Elution 1 containing purified gbr22 protein using 574 nm. Absorbance: 0.109 for n=1 and 0.111 for n=2.
Calculation of Elution 1 yield based on Nanodrop results:
280 nm: Average absorbance values for n=2 trials = 0.538
Beer’s Law: A = abc, a = 38850 M-1cm-1, b = 1 cm, A = 0 .538
c = 1.38 x 10-5 M (25794.2 gmol-1) = 0.36 mg/mL.
yield280 = 4.5 mL * 0.36 mg/mL = 1.606 mg.
574 nm: Average absorbance values for n=2 trials = 0.110
Beer’s Law: A = abc, a = 118300 M-1cm-1, b = 1 mm, A = 0.110
c = 9.30 x 10-6 M (25794.2 gmol-1) = 0.24 mg/mL.
yield280 = 4.5 mL * 0.24 mg/mL = 1.080 mg.
Figure 7: Dried SDS-PAGE gel results for protein characterization with lanes (from left to right): lane 1 contains a molecular weight standard, lanes 2 – 7 contains samples 1 – 6 from various points in the expression/purification process (see Materials and Methods section), and lanes 8 – 10 contains samples 4 – 6 from a different researcher.
Figure 8: Fermentas PageRuler pre-stained molecular weight standard as used in lane 1 of figure 7.
Discussion:
The purification process involved the use of lysozyme to digest the bacterial cell walls and benzonase to digest the DNA and RNA present in the lysate. Then, the HIS affinity tag system, which was incorporated into the fluorescent protein, served to bind the gbr22 to the nickel ions in the Ni-NTA Sample 1 contained the bacterial cell sample suspended in LB media and ampicillin, sample 2 contained lysed cell parts excluding the protein, sample 3 contained 1 M PBS, sample 4 contained 20 mM imidazole, 1 M PBS, and loosely bound proteins to the Ni-NTA resin, sample 5 contained 200 mM imidazole, 1M PBS, and a majority of the gbr22 protein, and sample 6 contained 200 mM imidazole, 1 M PBS, and the leftovers of the gbr22 protein. Sample 4 contained wash buffer (20 mM imidazole, 1 M PBS) which served to remove proteins that did not bind to the Ni-NTA. Sample 5 contained elution buffer (200 mM imidazole, 1 M PBS) which served to remove the Ni-NTA bound protein – gbr22. The relevant gel lanes in figure 7 are those that contained sample 5 because it theoretically contained the majority of the purified protein. S5* was well-purified because there is one dark band corresponding to the gbr22 protein (with a molecular weight of around 26 kDa according to figure 8) and only a few lighter bands. S5* has a purity of around 80%. S5 also contained a dark band at around 26 kDa, but it was not purified as effectively because it contained two other dark bands and least two lighter bands. S5 has a purity of around 30%. This suggests that there was either error in preparing the gel lane or in the column elution steps. Overall, though the empirically determined MW of gbr22 in both S5 and S5* are fairly accurate with regards to the accepted value of 25.8 kDa. The yield results based on the Nanodrop spectroscopy were fairly precise, with possible error in using the instrument due to contamination of the sample or a nonhomogenous sample.
Conclusions:
In this lab, gbr22 protein was expressed in E. Coli, purified, and characterized to determine the effectiveness of the expression and purification technique. It was found that the empirical technique was successful in extracting the gbr22 protein, but also had the potential of leaving unwanted proteins in the final sample. In addition, a yield on the order of 1 mg of protein could be obtained for a given colony of bacteria. In future VDS research, the process of utilizing bacteria to accurately harvest proteins is integral. Drug ligand candidates are very specific to which proteins they bind to, so it is important that the target protein be isolated in the presence of the ligand, else misleading results may be obtained. Knowing the procedure and being able to modify it at different steps is also important.
References:
[1] Nat Methods. 2008 Feb;5(2):135-46. Protein production and purification.
Introduction:
The process of obtaining protein for use in biochemical assays or antibody production involve protein expression and purification techniques [1]. The DNA coding for the protein must first be incorporated into a host cell which can range from bacterial to yeast cells. When using BL-21 bacterial E. Coli, a single recombinant colony is grown into a starter culture which is induced into protein expression and finally harvested for the target protein. Expression may be optimized by use of a tag (e.g. hexa-histidine affinity tag) or by varying expression induction conditions such as temperature and concentration [2]
The goal of protein purification is to isolate the protein. A clarified sample of soluble protein is first extracted via centrifuge and then clarified further by removing DNA and lipids. For His6-tags, a combination of imidazole buffer, phosphate buffer, and Ni-NTA resin is then used to bind and extract the protein [3].The success of expression and purification then can be gauged via gel electrophoresis and UV-vis spectroscopy.
Each individual protein has unique expression and purification protocols [1]. In this lab, gbr22, a purple protein found in coral, is expressed, purified, and characterized to determine the extent of purification. By utilizing appropriate expression and purification technique, characterization through gel electrophoresis should yield a molecular weight of gbr22 similar to the literature value as well as a high concentration of purified protein.
Materials & Methods:
gbr22 protein in the form of a plasmid with antibiotic resistance was mixed with a culture of bacteria and heat shocked to initiate transformation. The bacteria culture was then spread onto antibiotic plates incubated overnight at 37OC to yield purple colonies of living transformed bacteria. A starter culture was then grown with a single purple colony, LB media, and 100 mg/ml ampicillin and put into a shake incubator at 37OC for ~8 hours. The starter culture was then transferred into a larger culture with additional ampicillin and LB and shake incubated for 24 hours (sample 1 taken of large culture). The purple culture was then spun down to obtain a purple pellet of transformed cells. 1X PBS and 1 mg/mL lysozyme were added to the pellet and the final tube was frozen at -20OC.
The lysate was purified by benzonase to digest DNA, centrifuge (supernatant was taken as sample 2 at this point), and finally PES syringe filter of the clarified liquid lysate. The mixture was further purified via Ni-NTA affinity through a Bio-rad Econo column. 1X PBS, 20 mM imidazole, and 250 mM imidazole was run through the column to produce waste, wash, and two elutions. Samples 3, 4, 5, and 6 were taken of these steps, respectively. The Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) was then used to measure the absorbance of the first elution at 280 nm and 574 nm (the maximum absorbance wavelength of gbr-22).
In order to characterize the purified protein, gel electrophoresis was conducted with SDS-PAGE gel along with six samples taken at various points in the expression and purification steps (specified in the results section). The gel results were then compared to a molecular weight standard.
Results:
Calculation of Elution 1 yield based on Nanodrop results:
280 nm:
Average absorbance values for n=2 trials = 0.538
Beer’s Law: A = abc, a = 38850 M-1cm-1, b = 1 cm, A = 0 .538
c = 1.38 x 10-5 M (25794.2 gmol-1) = 0.36 mg/mL.
yield280 = 4.5 mL * 0.36 mg/mL = 1.606 mg.
574 nm:
Average absorbance values for n=2 trials = 0.110
Beer’s Law: A = abc, a = 118300 M-1cm-1, b = 1 mm, A = 0.110
c = 9.30 x 10-6 M (25794.2 gmol-1) = 0.24 mg/mL.
yield280 = 4.5 mL * 0.24 mg/mL = 1.080 mg.
Discussion:
The purification process involved the use of lysozyme to digest the bacterial cell walls and benzonase to digest the DNA and RNA present in the lysate. Then, the HIS affinity tag system, which was incorporated into the fluorescent protein, served to bind the gbr22 to the nickel ions in the Ni-NTA Sample 1 contained the bacterial cell sample suspended in LB media and ampicillin, sample 2 contained lysed cell parts excluding the protein, sample 3 contained 1 M PBS, sample 4 contained 20 mM imidazole, 1 M PBS, and loosely bound proteins to the Ni-NTA resin, sample 5 contained 200 mM imidazole, 1M PBS, and a majority of the gbr22 protein, and sample 6 contained 200 mM imidazole, 1 M PBS, and the leftovers of the gbr22 protein. Sample 4 contained wash buffer (20 mM imidazole, 1 M PBS) which served to remove proteins that did not bind to the Ni-NTA. Sample 5 contained elution buffer (200 mM imidazole, 1 M PBS) which served to remove the Ni-NTA bound protein – gbr22.
The relevant gel lanes in figure 7 are those that contained sample 5 because it theoretically contained the majority of the purified protein. S5* was well-purified because there is one dark band corresponding to the gbr22 protein (with a molecular weight of around 26 kDa according to figure 8) and only a few lighter bands. S5* has a purity of around 80%. S5 also contained a dark band at around 26 kDa, but it was not purified as effectively because it contained two other dark bands and least two lighter bands. S5 has a purity of around 30%. This suggests that there was either error in preparing the gel lane or in the column elution steps. Overall, though the empirically determined MW of gbr22 in both S5 and S5* are fairly accurate with regards to the accepted value of 25.8 kDa.
The yield results based on the Nanodrop spectroscopy were fairly precise, with possible error in using the instrument due to contamination of the sample or a nonhomogenous sample.
Conclusions:
In this lab, gbr22 protein was expressed in E. Coli, purified, and characterized to determine the effectiveness of the expression and purification technique. It was found that the empirical technique was successful in extracting the gbr22 protein, but also had the potential of leaving unwanted proteins in the final sample. In addition, a yield on the order of 1 mg of protein could be obtained for a given colony of bacteria.
In future VDS research, the process of utilizing bacteria to accurately harvest proteins is integral. Drug ligand candidates are very specific to which proteins they bind to, so it is important that the target protein be isolated in the presence of the ligand, else misleading results may be obtained. Knowing the procedure and being able to modify it at different steps is also important.
References:
[1] Nat Methods. 2008 Feb;5(2):135-46. Protein production and purification.
[2] European Molecular Biology Laboratory. Protein Expression and Purification Core Facility: Protein Expression. http://www.embl.de/pepcore/pepcore_services/protein_expression/index.html (accessed April 15 2012).
[3] European Molecular Biology Laboratory. Protein Expression and Purification Core Facility: Protein Expression. http://www.embl.de/pepcore/pepcore_services/protein_purification/index.html (accessed April 15 2012).